Arrays including a resin film and a patterned polymer layer

ABSTRACT

An example of an array includes a support, a cross-linked epoxy polyhedral oligomeric silsesquioxane (POSS) resin film on a surface of the support, and a patterned hydrophobic polymer layer on the cross-linked epoxy POSS resin film. The patterned hydrophobic polymer layer defines exposed discrete areas of the cross-linked epoxy POSS resin film, and a polymer coating is attached to the exposed discrete areas. Another example of an array includes a support, a modified epoxy POSS resin film on a surface of the support, and a patterned hydrophobic polymer layer on the modified epoxy POSS resin film. The modified epoxy POSS resin film includes a polymer growth initiation site, and the patterned hydrophobic polymer layer defines exposed discrete areas of the modified epoxy POSS resin film. A polymer brush is attached to the polymer growth initiation site in the exposed discrete areas.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/438,024, filed Dec. 22, 2016, the contents of which isincorporated by reference herein in its entirety.

BACKGROUND

Biological arrays are among a wide range of tools used to detect andanalyze molecules, including deoxyribonucleic acid (DNA) and ribonucleicacid (RNA). In these applications, the arrays are engineered to includeprobes for nucleotide sequences present in genes in humans and otherorganisms. In certain applications, for example, individual DNA and RNAprobes may be attached at small locations in a geometric grid (orrandomly) on an array support. A test sample, e.g., from a known personor organism, may be exposed to the grid, such that complementaryfragments hybridize to the probes at the individual sites in the array.The array can then be examined by scanning specific frequencies of lightover the sites to identify which fragments are present in the sample, byfluorescence of the sites at which the fragments hybridized.

Biological arrays may be used for genetic sequencing. In general,genetic sequencing involves determining the order of nucleotides ornucleic acids in a length of genetic material, such as a fragment of DNAor RNA. Increasingly longer sequences of base pairs are being analyzed,and the resulting sequence information may be used in variousbioinformatics methods to logically fit fragments together so as toreliably determine the sequence of extensive lengths of genetic materialfrom which the fragments were derived. Automated, computer-basedexamination of characteristic fragments have been developed, and havebeen used in genome mapping, identification of genes and their function,evaluation of risks of certain conditions and disease states, and soforth. Beyond these applications, biological arrays may be used for thedetection and evaluation of a wide range of molecules, families ofmolecules, genetic expression levels, single nucleotide polymorphisms,and genotyping.

SUMMARY

In some aspects is a composition comprising a support and a cross-linkedepoxy POSS resin film on a surface of the support. In some aspects thecomposition is suitable as an array for oligonucleotide sequencing or asa production intermediate. In some aspects, the resin film is patternedto define discrete areas within interstitial regions, and in someaspects, the discrete areas are wells. In other aspects, the compositioncomprises a hydrophobic polymer layer on the patterned resin film,including the discrete areas defined by the pattern (e.g., in the wells)and the interstitial regions. In other aspects, the hydrophobic polymerlayer is patterned to expose the resin film in the discrete areas orwells while remaining on the resin film in the interstitial areas of theresin film between the discrete areas or wells. In still other aspects,a polymer coating is attached to the patterned resin film in the exposeddiscrete areas of the cross-linked epoxy POSS resin film. Thecomposition may further comprise an amplification primer grafted to thepolymer coating. The cross-linked POSS resin film optionally comprises apolymer growth initiation site as described herein. In still otheraspects, a polymer brush is attached to the polymer growth initiationsite in the exposed discrete areas of the cross-linked epoxy POSS resinfilm.

In some aspects are methods of making the composition comprising asupport and a cross-linked epoxy POSS resin film on a surface of thesupport, comprising forming the cross-linked epoxy POSS resin film on asupport surface, where the forming involves mixing a support-bound epoxysilane with one or more epoxy-functionalized POSS reagents in thepresence of a photoacid generator and optionally a sensitizer to form asupport-bound resin precursor, and curing the resin precursor to form asupport-bound cross-linked epoxy POSS resin film. Such methods mayfurther comprise reacting a surface of a support with an epoxy silane toform the support-bound epoxy silane. In some aspects, the resin film ispatterned to define discrete areas within interstitial regions, and insome aspects, the discrete areas are wells. Such methods may furthercomprise forming a hydrophobic polymer layer on the cross-linked,support-bound epoxy POSS resin film on the support surface, wherein thehydrophobic polymer layer is patterned to expose the resin film in thediscrete areas or wells while remaining on the resin film in theinterstitial areas of the resin film between the discrete areas orwells.

In a first aspect is an array that includes a support, a cross-linkedepoxy polyhedral oligomeric silsesquioxane (POSS) resin film on asurface of the support, and a patterned hydrophobic polymer layer on thecross-linked epoxy POSS resin film, wherein the patterned hydrophobicpolymer layer defines exposed discrete areas of the cross-linked epoxyPOSS resin film, and a polymer coating is attached to the exposeddiscrete areas.

In some aspects are methods of forming arrays of this first aspect,which comprise forming a patterned hydrophobic polymer layer on across-linked epoxy POSS resin film on a support surface, therebyexposing discrete areas of the cross-linked epoxy POSS resin film. Thismethod may further comprise applying a polymer coating to form anattached coating portion on the exposed discrete areas and an unattachedcoating portion on the patterned hydrophobic layer; and washing theunattached coating portion off of the patterned hydrophobic layer. Themethod may further comprise forming the cross-linked epoxy POSS resinfilm on the support surface, the forming involving: mixing an epoxysilane and at least one epoxy POSS monomeric unit in the presence of aphotoacid generator and optionally a sensitizer to form a resinprecursor; depositing the resin precursor on the support surface; andcuring the resin precursor to form the cross-linked epoxy POSS resinfilm.

In a second aspect, an array includes a support, a modified epoxy POSSresin film on a surface of the support, and a patterned hydrophobicpolymer layer on the modified epoxy POSS resin film, where the patternedhydrophobic polymer layer defines exposed discrete areas of thecross-linked epoxy POSS resin film. In some instances, the modifiedepoxy POSS resin film includes a polymer growth initiation site, and thepatterned hydrophobic polymer layer defines exposed discrete areas ofthe modified epoxy POSS resin film. A polymer brush is attached to thepolymer growth initiation site in the exposed discrete areas. In somerespects, the array comprises a support, a modified epoxy polyhedraloligomeric silsesquioxane (POSS) resin film on a surface of the support,the modified epoxy POSS resin film including a polymer growth initiationsite, a patterned hydrophobic polymer layer on the modified epoxy POSSresin film, the patterned hydrophobic polymer layer defining exposeddiscrete areas of the modified epoxy POSS resin film, and a polymerbrush attached to the polymer growth initiation site in the exposeddiscrete areas.

Methods for producing arrays of the second aspect as described hereincomprise forming a patterned hydrophobic polymer layer on a modifiedepoxy polyhedral oligomeric silsesquioxane (POSS) resin film on asupport surface, thereby exposing discrete areas of the modified epoxyPOSS resin film. The modified epoxy POSS resin film includes a polymergrowth initiation site. In some aspects, a polymer brush is grown fromthe polymer growth initiation site in the exposed discrete areas. Thus,in some aspects, a second aspect of the method disclosed hereincomprises forming a patterned hydrophobic polymer layer on a modifiedepoxy polyhedral oligomeric silsesquioxane (POSS) resin film on asupport surface, thereby exposing discrete areas of the modified epoxyPOSS resin film, wherein the modified epoxy POSS resin film includes apolymer growth initiation site; and growing a polymer brush from thepolymer growth initiation site in the exposed discrete areas. In someaspects, the method further comprises forming the modified epoxy POSSresin film, where the forming involves mixing an epoxy silane, at leastone epoxy POSS monomeric unit, and an epoxy-functionalizedpolymerization agent (e.g., a radical polymerization agent, a cationicpolymerization agent, an anionic polymerization agent, a ring-openingmethathesis polymerization agent, or a controlled radical polymerizationagent) or controlled radical polymerization (CRP) agent in the presenceof a photoacid generator and an optional sensitizer to form a resinprecursor; depositing the resin precursor on the support surface; andcuring the resin precursor to form the modified epoxy POSS resin film.In some aspects, the at least one epoxy POSS monomeric unit isepoxycyclohexylalkyl POSS and glycidyl POSS.

BRIEF DESCRIPTION OF THE DRAWINGS

Features and advantages of examples of the present disclosure willbecome apparent by reference to the following detailed description anddrawings, in which like reference numerals correspond to similar, thoughperhaps not identical, components. For the sake of brevity, referencenumerals or features having a previously described function may or maynot be described in connection with other drawings in which they appear.

FIGS. 1A through 1F are cross-sectional views illustrating an example ofthe method disclosed herein, where FIG. 1E is an enlarged view of adepression of the array that is formed; and

FIGS. 2A through 2D are cross-sectional views illustrating anotherexample of the method disclosed herein, where FIG. 2C is an enlargedview of a depression of the array that is formed.

DETAILED DESCRIPTION

It is to be understood that any features of the first aspect of thearray may be combined together in any desirable manner. Moreover, it isto be understood that any combination of features of the first aspectand/or first method may be used together, and/or that any features fromeither or both of these aspects may be combined with any of the examplesdisclosed herein.

In some instances of the methods, compositions, and arrays describedherein, the cross-linked epoxy POSS resin film is patterned to definefeatures such as wells and intervening interstitial regions between thefeatures, and in other instances, the cross-linked epoxy POSS resin filmis not patterned. In instances in which the cross-linked epoxy POSSresin film is patterned, the pattern in the film defines features thatare the discrete portions that are also exposed by the pattern of thepatterned hydrophobic layer, e.g., the resin film comprises wells, andthe discrete regions exposed by the patterned hydrophobic layer are thewells in the film.

Examples of the method disclosed herein use the different epoxy POSSresin films in combination with the patterned hydrophobic layer toconfine where a polymer is applied or grows, or to allow forpreferential removal of the polymer from regions with the patternedhydrophobic layer over regions with exposed resin film. These methodseliminate the need for mechanical or chemical polymer removal processes,such as polishing, that are performed when the polymer is blanketlydeposited across the entire resin or solid support surface.

In one example of the method disclosed herein, a cross-linked epoxypolyhedral oligomeric silsesquioxane (POSS) resin film is used incombination with a patterned hydrophobic layer. The patternedhydrophobic layer exposes discrete portions of the cross-linked epoxyPOSS resin film, which serve as capture pads for a subsequently appliedpolymeric material, in part because the polymeric material is morehydrophilic than the patterned hydrophobic layer. The surface energy ofthe polymeric material is closer to the surface energy of thecross-linked epoxy POSS resin film than to the surface energy of thepatterned hydrophobic layer, and thus the polymeric material has betterwetting onto the cross-linked epoxy POSS resin film. In some instances,the resin film is chemically modified with capture groups that arecapable of forming covalent bonds with functional groups on thepolymeric material.

An example of this first aspect of the method further comprises formingthe cross-linked epoxy POSS resin film on the support surface, where theforming involves mixing an epoxy silane, epoxycyclohexylalkyl POSS, andglycidyl POSS in the presence of a photoacid generator and optionally asensitizer to form a resin precursor; depositing the resin precursor onthe support surface; and curing the resin precursor to form thecross-linked epoxy POSS resin film.

In this first aspect of the method, prior to forming the patternedhydrophobic polymer layer, the method can further comprise exposing thecross-linked epoxy POSS resin film to plasma aching or a chemicaltreatment to introduce —OH groups such as hydroxyl (C—OH or Si—OH)groups and/or carboxyl groups to the cross-linked epoxy POSS resin film.In some aspects, the method further comprises attaching functionalgroups to at least some of the —OH groups, the functional groups beingselected from the group consisting of:

wherein n ranges from 1 to 20,

and wherein - - - represents an alkylsilane (e.g., by reaction of thehydroxyl groups with a trialkoxyalkylsilane), a poly(ethyleneglycol)-silane (e.g., by reaction of the hydroxyl groups with atrialkoxysilane poly(ethylene glycol)), an alkyl (e.g., by reaction ofthe hydroxyl groups with an alkyl halide), or a polyethylene glycolchain.

Another example of this first aspect of the method further comprisesforming the cross-linked epoxy POSS resin film on the support surface,where the forming involves mixing, in the presence of an optionalsensitizer and a photoacid generator, an epoxy silane,epoxycyclohexylalkyl POSS, glycidyl POSS, and a POSS core including atleast one epoxy functional group and a non-epoxy functional group toform a resin precursor; depositing the resin precursor on the supportsurface; and curing the resin precursor to form the cross-linked epoxyPOSS resin film.

In another example of this first aspect, the method comprises formingthe cross-linked epoxy POSS resin film on the support surface, where theforming involves mixing, in the presence of a photoacid generator andoptionally a sensitizer, a support-bound epoxy silane with one or moreepoxy-functionalized POSS reagents and a POSS core including one epoxyfunctional group and a non-epoxy functional group to form asupport-bound resin precursor, and curing the resin precursor to form asupport-bound cross-linked epoxy POSS resin film.

In an example of this first aspect of the method, washing involvessonication in water. In another example, washing involves dunk washingand spraying or mechanical scrubbing.

In an example of this first aspect of the method, forming the patternedhydrophobic polymer layer involves i) depositing a hydrophobic polymeron the cross-linked epoxy POSS resin film and patterning the depositedhydrophobic polymer using at least one of nanoimprint lithography andphotolithography; or ii) depositing the hydrophobic polymer in a patternon the cross-linked epoxy POSS resin film using patterned printing, forexample, at least one of inkjet printing and microcontact printing, oraerosol patterned printing.

In examples of this first aspect of the method further comprisesgrafting an amplification primer to the attached coating portion.Examples of the first aspect of the array further comprise amplificationprimers grafted to the attached coating portion.

In these first aspects of the method and array, the patternedhydrophobic layer is selected from the group consisting of afluoropolymer, a negative tone photoresist, and a polysiloxane.

In another example of the method disclosed herein, a modified epoxy POSSresin film is used in combination with a patterned hydrophobic layer.The patterned hydrophobic layer exposes discrete portions of themodified epoxy POSS resin film. The modified epoxy POSS resin filmincludes an epoxy-functionalized controlled radical polymerization (CRP)agent, which acts as an initiator species for polymer growth. Thepatterned hydrophobic layer confines the polymer growth to the discreteportions.

In some examples, the polymerization agent or CRP agent is a POSS coreincluding at least one epoxy functional group and a polymerization agentor CRP agent functional group. In some examples, theepoxy-functionalized CRP agent is an epoxy-functionalized reversibleaddition-fragmentation chain transfer (RAFT) agent or anepoxy-functionalized atom transfer radical polymerization (ATRP)initiator. In certain examples, a molar or mass ratio ofepoxycyclohexylalkyl POSS and glycidyl POSS to epoxy-functionalized CRPagent ranges from about 1:1 to about 9:1.

Another example of this second aspect of the method comprises forming across-linked epoxy POSS resin film on a support surface, where theforming involves mixing a support-bound epoxy silane with one or moreepoxy-functionalized POSS reagents in the presence of anepoxy-functionalized controlled radical polymerization (CRP) agent, aphotoacid generator, and optionally a sensitizer, to form asupport-bound resin precursor, and curing the resin precursor to form asupport-bound cross-linked epoxy POSS resin film. Such examples mayfurther comprise reacting a surface of a support with an epoxy silane toform the cross-linked, support-bound epoxy silane. Such methods mayfurther comprise forming a patterned hydrophobic polymer layer on thecross-linked, support-bound epoxy POSS resin film on the support surfaceas described herein.

In this second aspect of the method, prior to forming the patternedhydrophobic polymer layer, the method can further comprise exposing thecross-linked epoxy POSS resin film to plasma ashing or a chemicaltreatment to introduce —OH groups (e.g., hydroxyl (C—OH, Si—OH) and/orcarboxyl) groups to the cross-linked epoxy POSS resin film; andattaching functional groups or CRP agents to at least some of thehydroxyl groups, the functional groups being selected from the groupconsisting of:

wherein n ranges from 1 to 20,

and wherein - - - represents an alkylsilane (e.g., by reaction of thehydroxyl groups with a trialkoxyalkylsilane), a poly(ethyleneglycol)-silane (e.g., by reaction of the hydroxyl groups with atrialkoxysilane poly(ethylene glycol)), an alkyl (e.g., by reaction ofthe hydroxyl groups with an alkyl halide), or a polyethylene glycolchain.

Another example of this second aspect of the method comprises formingthe modified epoxy POSS resin film, where the forming involves mixing,in the presence of an optional sensitizer and a photoacid generator, anepoxy silane, epoxycyclohexylalkyl POSS, glycidyl POSS, and a POSS coreincluding at least one epoxy functional group and a non-epoxy functionalgroup to form a resin precursor; depositing the resin precursor on thesupport surface; curing the resin precursor to form an initiallymodified epoxy POSS resin film; and introducing a controlled radicalpolymerization (CRP) agent functional group to the initially modifiedepoxy POSS resin film to form the modified epoxy POSS resin film. Thenon-epoxy functional group is (a) a reactive group that is orthogonallyreactive to an epoxy group (i.e., reacts under different conditions thanan epoxy group), that serves as a handle for coupling the resin to anamplification primer, a polymer, or a polymerization agent; or (b) agroup that adjusts the mechanical or functional properties of the resin,e.g., surface energy adjustments. In some aspects, the non-epoxyfunctional group being selected from the group consisting of an azide, athiol, a poly(ethylene glycol), a norbornene, and a tetrazine. In otheraspects, the non-epoxy functional group is an amino, hydroxyl, alkynyl,ketone, aldehyde, or ester group. In other aspects, the non-epoxyfunctional group is an alkyl, aryl, alkoxy, or haloalkyl group.

In an example of the second aspect of the method, forming the patternedhydrophobic polymer layer involves i) depositing a hydrophobic polymeron the modified epoxy POSS resin film and patterning the depositedhydrophobic polymer using at least one of nanoimprint lithography andphotolithography; or ii) depositing the hydrophobic polymer in a patternon the modified epoxy POSS resin film using patterned printing such asat least one of inkjet printing and microcontact printing, or aerosolpatterned printing.

It is to be understood that any features of the second aspect of themethod may be combined together in any desirable manner. Moreover, it isto be understood that any combination of features of the first aspect ofthe method and/or array and/or the second aspect of the method may beused together, and/or that any features from any of these aspects may becombined with any of the examples disclosed herein.

In some examples of the second aspect of the array and method, thepolymer brush is a copolymer, such as a random, ordered, or blockcopolymer. In some aspects, the polymer brush is further functionalizedby radical exchange. Functionalization of attached polymer networks maybe performed using reactive units that mediate C—H insertion reactions,such as aromatic carbonyl compounds (diphenylketone derivatives), azocompounds, sulfonyl azides, aryl azides, and aziridines.

An example of this second array further comprises an amplificationprimer grafted to the polymer brush. An example of this second methodfurther comprises grafting an amplification primer to the polymer brush.

In these second aspects of the method and array, the patternedhydrophobic layer is selected from the group consisting of afluoropolymer, a negative tone photoresist, and a polysiloxane.

It is to be understood that any features of the second aspect of thearray may be combined together in any desirable manner. Moreover, it isto be understood that any combination of features of the first aspect ofthe method and/or array and/or the second aspect of the method and/orarray may be used together, and/or that any features from any of theseaspects may be combined with any of the examples disclosed herein.

In some aspects of the methods, arrays, and compositions describedherein, the polymer coating includes a recurring unit of Formula (I):

-   wherein:-   R¹ is H or optionally substituted alkyl;-   R^(A) is selected from the group consisting of azido, optionally    substituted amino, optionally substituted alkenyl, optionally    substituted hydrazone, optionally substituted hydrazine, carboxyl,    hydroxy, optionally substituted tetrazole, optionally substituted    tetrazine, nitrile oxide, nitrone, and thiol;-   R⁵ is selected from the group consisting of H and optionally    substituted alkyl;-   each of the —(CH₂)_(p)— can be optionally substituted;-   p is an integer in the range of 1 to 50;-   n is an integer in the range of 1 to 50,000; and-   m is an integer in the range of 1 to 100,000.

In the structure of Formula (I), one of ordinary skill in the art willunderstand that the “n” and “m” subunits are recurring subunits that arepresent in a random order throughout the polymer.

A particular example of a polymer coating ispoly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide, PAZAM (seefor example, U.S. Patent Publication Nos. 2014/0079923 A1, or2015/0005447 A1, each of which is incorporated herein by reference inits entirety), which comprises the structure shown below:

wherein n is an integer in the range of 1-20,000, and m is an integer inthe range of 1-100,000. As with Formula (I), one of ordinary skill inthe art will recognize that the “n” and “m” subunits are recurring unitsthat are present in random order throughout the polymer structure.

The molecular weight of the Formula (I) or PAZAM polymer may range fromabout 10 kDa to about 1500 kDa, or may be, in a specific example, about312 kDa.

In some examples, the Formula (I) or PAZAM polymer is a linear polymer.In some other examples, the Formula (I) or PAZAM polymer is a lightlycross-linked polymer. In other examples, the Formula (I) or PAZAMpolymer comprises branching.

Other examples of suitable polymer materials include those having acolloidal structure, such as agarose; or a polymer mesh structure, suchas gelatin; or a cross-linked polymer structure, such as polyacrylamidepolymers and copolymers, silane free acrylamide (SFA, see, for example,U.S. Patent Publication No. 2011/0059865, which is incorporated hereinby reference in its entirety), or an azidolyzed version of SFA. Examplesof suitable polyacrylamide polymers may be formed from acrylamide and anacrylic acid or an acrylic acid containing a vinyl group as described,for example, in WO 2000/031148 (incorporated herein by reference in itsentirety) or from monomers that form [2+2] photo-cycloadditionreactions, for example, as described in WO 2001/001143 or WO2003/0014392 (each of which is incorporated herein by reference in itsentirety). Other suitable polymers are co-polymers of SFA and SFAderivatized with a bromo-acetamide group (e.g., BRAPA), or co-polymersof SFA and SFA derivatized with an azido-acetamide group.

It is to be understood that terms used herein will take on theirordinary meaning in the relevant art unless specified otherwise. Severalterms used herein and their meanings are set forth below.

The singular forms “a”, “an”, and “the” include plural referents unlessthe context clearly dictates otherwise.

The terms comprising, including, containing and various forms of theseterms are synonymous with each other and are meant to be equally broad.

As used herein, an “acrylate” refers to a “CH₂═CHCOO—” functional group(i.e.,

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that is fully saturated (i.e., contains no double or triplebonds). The alkyl group may have 1 to 20 carbon atoms. Example alkylgroups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tertiary butyl, pentyl, hexyl, and the like. As an example, thedesignation “C1-4 alkyl” indicates that there are one to four carbonatoms in the alkyl chain, i.e., the alkyl chain is selected from thegroup consisting of methyl, ethyl, propyl, iso-propyl, n-butyl,isobutyl, sec-butyl, and t-butyl.

The alkyl may be substituted with a halide or halogen, which means anyone of the radio-stable atoms of column 7 of the Periodic Table of theElements, e.g., fluorine, chlorine, bromine, or iodine. This group isreferred to as an “alkyl halide”.

The alkyl may also be singular bonded to an oxygen atom. This group isan “alkoxy”. An example of an alkoxy is a hydroxyl terminated ethoxy(i.e.,

wherein n ranges from 1 to 20). This group may also be referred to ashydroxyl terminated poly(ethylene glycol).

As used herein, “alkenyl” refers to a straight or branched hydrocarbonchain containing one or more double bonds. The alkenyl group may have 2to 20 carbon atoms. Example alkenyl groups include ethenyl, propenyl,butenyl, pentenyl, hexenyl, and the like. The alkenyl group may bedesignated as, for example, “C2-4 alkenyl,” which indicates that thereare two to four carbon atoms in the alkenyl chain.

As used herein, “alkynyl” refers to a straight or branched hydrocarbonchain containing one or more triple bonds (e.g.,

The alkynyl group may have 2 to 20 carbon atoms. The alkynyl group maybe designated, for example, as “C2-4 alkynyl,” which indicates thatthere are two to four carbon atoms in the alkynyl chain.

An “amino” functional group refers to an —NR_(a)R_(b) group, where R_(a)and R_(b) are each independently selected from hydrogen (e.g.,

C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, C6-10 aryl,5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as definedherein.

As used herein, “aryl” refers to an aromatic ring or ring system (i.e.,two or more fused rings that share two adjacent carbon atoms) containingonly carbon in the ring backbone. When the aryl is a ring system, everyring in the system is aromatic. The aryl group may have 6 to 18 carbonatoms, which may be designated as C6-18. Examples of aryl groups includephenyl, naphthyl, azulenyl, and anthracenyl.

As used herein, the term “attached” refers to the state of two thingsbeing joined, fastened, adhered, connected or bound to each other. Forexample, a nucleic acid can be attached to a polymer coating by acovalent or non-covalent bond. A covalent bond is characterized by thesharing of pairs of electrons between atoms. A non-covalent bond is achemical bond that does not involve the sharing of pairs of electronsand can include, for example, hydrogen bonds, ionic bonds, van der Waalsforces, hydrophilic interactions and hydrophobic interactions.

An “azide” or “azido” functional group refers to —N₃ (e.g.,

As used herein, “carbocyclyl” means a ring or ring system containingcarbon atoms in the ring backbone. When the carbocyclyl is a ringsystem, two or more rings may be joined together in a fused, bridged orspiro-connected fashion. Carbocyclyls may have any degree of saturation,provided that at least one ring in a ring system is not aromatic. Thus,carbocyclyls include cycloalkyls, cycloalkenyls, and cycloalkynyls. Thecarbocyclyl group may have 3 to 20 carbon atoms (i.e., C3-20).

As used herein, “curing” means treatment of a polymer or resin precursorto promote polymerization and cross-linking. With respect to the POSSresin film described herein, curing refers to polymerization andcross-linking of the POSS resin precursors and/or components. Curing maybe accomplished under a variety of conditions, such as exposure toactinic radiation, such as visible light radiation or ultraviolet (UV)radiation, or radiation of a wavelength between about 240 and 380 nm,and/or elevated temperature. Curing radiation may be provided by an Hglamp. Suitable curing temperatures may range from about 20° C. to about80° C. In some instances, curing may be completed using exposure to hardbake conditions that help drive the cross-linking reaction to completion(e.g., UV initiates the polymerization/cross-linking process and thereaction continues in the dark until complete). In some instances, ahard bake also dries or dehydrates the cross-linked epoxy POSS resinfilm to drive out any solvent(s) that may remain after curing. Suitablehard bake temperatures include temperatures from about 100° C. to about300° C. An example of a device that can be used for hard baking includesa hot plate.

As used herein, “cycloalkyl” means a fully saturated carbocyclyl ring orring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

As used herein, “cycloalkylene” means a fully saturated carbocyclyl ringor ring system that is attached to the rest of the molecule via twopoints of attachment.

As used herein, “cycloalkenyl” or “cycloalkene” means a carbocyclyl ringor ring system having at least one double bond, wherein no ring in thering system is aromatic. Examples include cyclohexenyl or cyclohexeneand norbornene or norbornyl (e.g.,

Also as used herein, “heterocycloalkenyl” or “heterocycloalkene” means acarbocyclyl ring or ring system with at least one heteroatom in ringbackbone, having at least one double bond, wherein no ring in the ringsystem is aromatic.

As used herein, “cycloalkynyl” or “cycloalkyne” means a carbocyclyl ringor ring system having at least one triple bond, wherein no ring in thering system is aromatic. An example is cyclooctyne (e.g.,

Another example is bicyclononyne (i.e., a bicyclic ring system, such as

Also as used herein, “heterocycloalkynyl” or “heterocycloalkyne” means acarbocyclyl ring or ring system with at least one heteroatom in ringbackbone, having at least one triple bond, wherein no ring in the ringsystem is aromatic.

As used herein, the term “carboxylic acid” or “carboxyl” as used hereinrefers to —C(O)OH.

The term “depositing,” as used herein, refers to any suitableapplication technique, which may be manual or automated. Generally,depositing may be performed using vapor deposition techniques, coatingtechniques, grafting techniques, or the like. Some specific examplesinclude chemical vapor deposition (CVD), plasma-enhanced CVD, initiatedCVD, metal-organic CVD, spray coating, spin coating, dunk or dipcoating, puddle dispensing, inkjet printing, screen printing, ormicrocontact printing.

As used herein, the term “depression” refers to a discrete concavefeature, defined by the patterned hydrophobic layer, having a surfaceopening that is completely surrounded by interstitial region(s) of thepatterned hydrophobic layer. Depressions can have any of a variety ofshapes at their opening in a surface including, as examples, round,elliptical, square, polygonal, star shaped (with any number ofvertices), etc. The cross-section of a depression taken orthogonallywith the surface can be curved, square, polygonal, hyperbolic, conical,angular, etc. As examples, the depression can be a well or a flowchannel.

The term “each,” when used in reference to a collection of items, isintended to identify an individual item in the collection, but does notnecessarily refer to every item in the collection. Exceptions can occurif explicit disclosure or context clearly dictates otherwise.

The term “epoxy” as used herein refers to

As used herein, “heteroaryl” refers to an aromatic ring or ring system(i.e., two or more fused rings that share two adjacent atoms) thatcontain(s) one or more heteroatoms, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen and sulfur, inthe ring backbone. When the heteroaryl is a ring system, every ring inthe system is aromatic. The heteroaryl group may have 5-18 ring members.

As used herein, “heterocyclyl” means a non-aromatic cyclic ring or ringsystem containing at least one heteroatom in the ring backbone.Heterocyclyls may be joined together in a fused, bridged orspiro-connected fashion. Heterocyclyls may have any degree of saturationprovided that at least one ring in the ring system is not aromatic. Inthe ring system, the heteroatom(s) may be present in either anon-aromatic or aromatic ring. The heterocyclyl group may have 3 to 20ring members (i.e., the number of atoms making up the ring backbone,including carbon atoms and heteroatoms). The heterocyclyl group may bedesignated as “3-6 membered heterocyclyl” or similar designations. Insome examples, the heteroatom(s) are O, N, or S.

The term “hydrazine” or “hydrazinyl” as used herein refers to a —NHNH₂group.

As used herein, the term “hydrazone” or “hydrazonyl” as used hereinrefers to a

group in which R_(a) and R_(b) are previously defined herein.

As used herein, “hydroxyl” is an —OH group. Hydroxyl groups as describedherein may be attached to carbon or silicon atoms.

As used herein, the term “interstitial region” refers to an area of thepatterned hydrophobic polymer layer that separates exposed areas of anunderlying resin film. An interstitial region can separate one featurethat is defined by the patterned hydrophobic polymer layer (e.g., adepression) from another feature that is defined by the patternedhydrophobic polymer layer. The two features that are separated from eachother can be discrete, i.e., lacking contact with each other. In anotherexample, an interstitial region can separate a first portion of afeature from a second portion of a feature. In many examples, theinterstitial region is continuous whereas the features are discrete, forexample, as is the case for a plurality of wells defined in an otherwisecontinuous patterned hydrophobic polymer layer. The separation providedby an interstitial region can be partial or full separation.Interstitial regions have the hydrophobic polymer layer as a surfacematerial, and the features defined by the hydrophobic polymer layer havethe resin film as a surface material. The term “interstitial region” isalso used herein where the resin film itself is patterned, to refer to aregion that separates one feature defined by the patterned film fromanother feature defined by the patterned film.

An “N-amido” group refers to a “—N(R_(a))C(═O)R_(b)” group in whichR_(a) and R_(b) are each independently selected from hydrogen, C1-6alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, C6-10 aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein).An example of an N-amido group is

wherein R_(a) is hydrogen and R_(b) is a C2 alkenyl. This particularN-amido is also an acrylamide. It is to be understood that the H atom inthe acrylamide could be replaced with an alkyl or another functionalgroup, and thus a substituted acrylamide may be used. Moreover, R_(b)could be an alkyl substituted C2 alkenyl (yielding, e.g., amethacrylamide group).

“Nitrile oxide,” as used herein, means a “R_(a)C≡N⁺O⁻” group in whichR_(a) is previously defined herein. Examples of preparing nitrile oxideinclude in situ generation from aldoximes by treatment with chloramide-Tor through action of base on imidoyl chlorides [RC(Cl)═NOH].

“Nitrone,” as used herein, means a “R_(a)R_(b)C═NR_(c) ⁺O⁻” group inwhich R_(a) and R_(b) are previously defined herein and R_(c) isselected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl,C6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

As used herein, a “nucleotide” includes a nitrogen containingheterocyclic base, a sugar, and one or more phosphate groups.Nucleotides are monomeric units of a nucleic acid sequence. In RNA, thesugar is a ribose, and in DNA a deoxyribose, i.e. a sugar lacking ahydroxyl group that is present at the 2′ position in ribose. Thenitrogen containing heterocyclic base (i.e., nucleobase) can be a purinebase or a pyrimidine base. Purine bases include adenine (A) and guanine(G), and modified derivatives or analogs thereof. Pyrimidine basesinclude cytosine (C), thymine (T), and uracil (U), and modifiedderivatives or analogs thereof. The C-1 atom of deoxyribose is bonded toN-1 of a pyrimidine or N-9 of a purine.

As used herein, the term “photoacid generator” is a compound thatbecomes more acidic or releases proton ions upon absorption of light.Exemplary photoacid generators include iodonium salts such asbis(4-tert-butylphenyl)iodonium perfluoro-1-butanesulfonate orbis-(4-tert-butylphenyl)iodonium p-toluenesulfonate, and sulfoniumtriflate compounds such as (4-tert-butylphenyl)diphenylsulfoniumtriflate, or triphenylsulfonium triflate. In alternative embodiments,curing could be performed under thermal conditions, with reagents thatrelease strong acid in situ upon exposure to heat.

As used herein, “plasma ashing” refers to a process of removing organicmatter from a patterned wafer or surface (e.g., a resin film) by anoxygen plasma or an air plasma. The products that result from plasmaashing may be removed with a vacuum pump/system. Plasma ashing canactivate a support surface by introducing reactive —OH or hydroxylgroups. Introduced hydroxyl groups can be bound to, e.g., carbon and/orsilicon atoms in the resin film. Introduced groups may also includecarboxyl groups.

As used herein, the terms “polymer coating” and “polymer brush” areintended to mean a semi-rigid polymeric material that is permeable toliquids and gases and that is tethered to the substrate/support. Thepolymer coating and polymer brush may be a hydrogel that can swell whenliquid is taken up and can contract when liquid is removed by drying.The polymer coating may be deposited, and the polymer brush may be grownfrom a polymer growth initiation site.

As used herein, the term “polyhedral oligomeric silsesquioxane” (POSS)refers to a chemical composition that is a hybrid intermediate(RSiO_(1.5)) between that of silica (SiO₂) and silicone (R₂SiO). Thecomposition is an organosilicon compound with the chemical formula[RSiO_(3/2)]_(n), where the R groups can be the same or different. Thecomposition may comprise one or more different cage or core structuresas monomeric units. In some instances, the structure comprises apolyoctahedral cage or core structure. For example, the polyhedralstructure may be a T₈ structure, such as:

and represented by:

This monomeric unit typically has eight arms of functional groups R₁through R₈.

The monomeric unit may have a cage structure with 10 silicon atoms and10 R groups, referred to as T₁₀, such as:

or may have a cage structure with 12 silicon atoms and 12 R groups,referred to as T₁₂, such as:

The POSS material may comprise T₆, T₁₄, or T₁₆ cage structures. Theaverage cage content can be adjusted during the synthesis, and/orcontrolled by purification methods, and a distribution of cage sizes ofthe monomeric unit(s) may be used in the examples disclosed herein. Asexamples, any of the cage structures may be present in an amount rangingfrom about 30% to about 100% of the total POSS monomeric units used. ThePOSS material may be a mixture of cage structures along with open andpartially open cage structures. Thus, the POSS resin precursors andresins described herein comprise epoxy POSS materials, which may be amixture of silsesquioxane configurations. For example, any POSS materialdescribed herein may be a mixture of discrete POSS cages andnon-discrete silsesquioxane structures and/or incompletely condensed,discrete structures, such as polymers, ladders, and the like. Thepartially condensed materials would therefore include epoxy R groups asdescribed herein at some silicon vertices, but some silicon atoms wouldnot be substituted with the R groups and could be substituted insteadwith OH groups. In some examples, the POSS materials comprise a mixtureof various forms, such as:

In the examples disclosed herein, at least one of R₁ through R₈ or R₁₀or R₁₂ comprises an epoxy, and thus the POSS is referred to as an epoxyPOSS. In some examples, a majority of the arms, such as the eight, ten,or twelve arms, or R groups, comprise epoxy groups. In other examples,R₁ through R₈ or R₁₀ or R₁₂ are the same, and thus each of R₁ through R₈or R₁₀ or R₁₂ comprises an epoxy group. Throughout this disclosure, thistype of POSS (i.e., in which R₁ through R₈ or R₁₀ or R₁₂ comprise thesame epoxy group) may be represented by the word “POSS” with aparticular epoxy functional group shown attached to the POSS. Forexample,

is the POSS cage with an epoxycyclohexylmethyl functional group as eachof R₁ through R₈ or R₁₀ or R₁₂. In other examples, R₁ through R₈ or R₁₀or R₁₂ are not the same, and thus at least one of R₁ through R₈ or R₁₀or R₁₂ comprises epoxy and at least one other of R₁ through R₈ or R₁₀ orR₁₂ is a non-epoxy functional group, which in some cases is selectedfrom the group consisting of an azide/azido, a thiol, a poly(ethyleneglycol), a norbornene, and a tetrazine, or further, for example, alkyl,aryl, alkoxy, and haloalkyl groups. In some aspects, the non-epoxyfunctional group is selected to increase the surface energy of theresin. In these other examples, the ratio of epoxy groups to non-epoxygroups ranges from 7:1 to 1:7, or 9:1 to 1:9, or 11:1 to 1:11. In any ofthe examples, disubstituted or monosubstituted (terminal) epoxy group(s)allow the monomeric unit to polymerize into a cross-linked matrix (i.e.,resin film) upon initiation using ultraviolet (UV) light and an acid. Insome aspects, the epoxy POSS comprises terminal epoxy groups.

When the epoxy POSS is referred to as a “modified epoxy POSS,” it ismeant that a controlled radical polymerization (CRP) agent and/oranother functional group of interest is incorporated into the resin orcore or cage structure as one or more of the functional group R₁ throughR₈ or R₁₀ or R₁₂. Similarly, when the epoxy POSS resin film is referredto as a “modified epoxy POSS resin film,” it is meant that a controlledradical polymerization (CRP) agent and/or another functional group ofinterest is incorporated into the cross-linked matrix.

As used herein, the “primer” is defined as a single stranded nucleicacid sequence (e.g., single strand DNA or single strand RNA) that servesas a starting point for DNA or RNA synthesis. The 5′ terminus of theprimer may be modified to allow a coupling reaction with the coatinglayer of the functionalized molecule. The primer length can be anynumber of bases long and can include a variety of non-naturalnucleotides. In an example, the sequencing primer is a short strand,including from 10 to 60 bases.

As used herein, the term “sensitizer” refers to a reagent that promotesphotoreactivity of component monomers by release of a reactive speciessuch as a free radical, e.g., a photoinitiator, a free radicalinitiator, azobisisobutyronitrile (AIBN), benzoyl peroxide,1-hydroxycyclohexyl phenyl ketone (HCPK), or a thioxanthenone. In someaspects, the sensitizer is selected to provide improved energy matchingwith the photoacid generator such that acid is released from thephotoacid generator under the selected uv conditions.

As used herein, the term “silane” refers to an organic or inorganiccompound containing one or more silicon atoms. An example of aninorganic silane compound is SiH₄, or halogenated SiH₄ where hydrogen isreplaced by one or more halogen atoms. An example of an organic silanecompound is X—R^(B)—Si(OR^(C))₃, wherein X is a functionalizable organicgroup, such as amino, methacrylate, thiol, alkyl, alkenyl, cycloalkenyl,alkynyl, or epoxy, which can be used to bond with a surface and/or apolymer; R^(B) is a spacer, for example an alkylene, heteroalkylene, or—(CH₂)_(n)—, wherein n is 0 to 1000 or 1 to 100 or 1 to 10 or 2 to 6;R^(C) is selected from hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted aryl,optionally substituted 5-10 membered heteroaryl, and optionallysubstituted 5-10 membered heterocyclyl, as defined herein. In someinstances, each R^(C) is the same, and in others, they may be different.In some examples, X is alkenyl or cycloalkenyl, R^(B) is —(CH₂)_(n)—,wherein n is 2 to 6, and/or R^(C) is alkyl. In another example, a silanecompound is X—R^(B)—Si(R^(D))₃, where X and R^(B) are as defined above,and each R^(D) is independently R^(C) or OR^(C). In some examples, Xcomprises a substrate or support. Generally, the alkoxysilane moiety isused to condense with —OH groups such as on the surface of a metal oxideor plasma-treated epoxy POSS network. The X functional group isorthogonal to the alkoxysilane, and is used to separately couple with aCRP or other initiator. The orthogonal nature of the reactive groupsallows for incorporation of the CRP unit after the POSS resin curingstep, or after a silanization step of the cross-linked resin. As usedherein, the term “silane” can include mixtures of different silaneand/or silane derivative compounds.

The terms “substrate” and “support” are used interchangeably herein, andrefer to a material on which a resin film is deposited. Examples ofsuitable supports include glass and modified or functionalized glass,plastics (including acrylics, polystyrene and copolymers of styrene andother materials, poly(vinyl chloride), polyesters, polycarbonates,poly(methyl methacrylate), polypropylene, polyethylene, polybutylene,polyurethanes, polytetrafluoroethylene (PTFE) (such as TEFLON® fromChemours), cyclic olefins/cyclo-olefin polymers (COP) or copolymers(COC) (such as ZEONOR® from Zeon), polyimides, etc.), nylon, ceramics,silica, fused silica, or other silica-based materials, silicon andmodified silicon, silicon dioxide, silicon nitride, silicon hydride,carbon, metals, inorganic glasses, and optical fiber bundles. Whileseveral examples have been provided, it is to be understood that anyother suitable substrate/support may be used.

The term “surface chemistry,” as used herein refers to a polymer coatingor polymer brush and primer(s) attached to at least a portion of anepoxy POSS resin film on a surface of a support/substrate.

A “thiol” functional group refers to —SH (e.g.,

As used herein, the terms “tetrazine” and “tetrazinyl” refer tosix-membered heteroaryl group including four nitrogen atoms. Tetrazinecan be optionally substituted. In an example, tetrazine is part ofmulti-ring structure where the rings do not share carbon atoms (e.g.,

“Tetrazole,” as used herein, refer to five-membered heterocyclic groupincluding four nitrogen atoms.

The term “wetting agent,” as used herein, refers to an additive thataids in surface coverage by components of the resin precursor mixture.Examples include surfactants, such as polyacrylate surfactants orsilicone surfactants.

As used herein, the term “YES method” refers a chemical vapor depositionprocess developed by Illumina, Inc. which uses the chemical vapordeposition tool provided by Yield Engineering Systems (“YES”). The toolincludes three different vapor deposition systems. The automatedYES-VertaCoat silane vapor system is designed for volume production witha flexible wafer handling module that can accommodate 200 mm or 300 mmwafers. The manual load YES-1224P Silane Vapor System is designed forversatile volume production with its configurable large capacitychambers. Yes-LabKote is a low-cost, tabletop version that is ideal forfeasibility studies and for R&D.

The aspects and examples set forth herein and recited in the claims canbe understood in view of the above definitions.

FIGS. 1A through 1F together illustrate an example of the methoddisclosed herein, which forms an example of the array disclosed herein.FIG. 1E is an enlarged view of a depression of the array that is formed.

FIG. 1A illustrates a support 12 with a cross-linked epoxy POSS resinfilm 14 formed thereon. Any example of the support 12 previouslydescribed herein may be used. In an example, the support 12 with thecross-linked epoxy POSS resin film 14 formed thereon is commerciallyavailable. In another example, the cross-linked epoxy POSS resin film 14is formed on the support 12.

Generally, the cross-linked epoxy POSS resin film 14 may be formed byforming a resin precursor, depositing the resin precursor on a surfaceof the support 12, and irradiating UV light to cure the resin precursorand to form the cross-linked epoxy POSS resin film 14.

The resin precursor is a mixture, which includes at least an epoxy POSSmonomeric unit. The precursor comprises silicon-containing moieties suchas D-silicons (attached to two oxygens), T-silicons (attached to 3oxygens), and Q-silicons (attached to 4 oxygens). As discussed above,the POSS materials may comprise cage polyhedral structures, discrete butincompletely condensed polyhedral structures, or non-discretesilsesquioxane structures, each of varying size. Examples of the epoxyPOSS monomeric units include epoxycyclohexyl alkyl POSS (where the alkylis a linker between the POSS cage and the epoxycyclohexyl groups, and ismethyl, ethyl, etc.), glycidyl POSS (where the R₁-R₈ or R₁₀ or R₁₂groups include an alkyl (e.g., methyl, ethyl, propyl, etc.) attached toa glycidyl ether; e.g.,

octaglycidyldimethylsilyl POSS, or the like. In some examples, the resinprecursor includes one type of epoxy POSS monomeric unit. In otherexamples, the resin precursor includes different epoxy POSS monomericunits. When two different epoxy POSS monomeric units are used incombination, any suitable mass or molar ratio of the two units may beselected. For example, a first epoxy POSS monomeric unit may be presentin an amount (X) ranging from about 10 mol % to about 90 mol % of thetotal amount of epoxy POSS monomeric units and a second epoxy POSSmonomeric unit may make up the balance of the total monomeric units(i.e., 100 mol %-X mol %). In an example, the epoxycyclohexylalkyl POSSand glycidyl POSS are used together in a mass or molar ratio of about3:1, although, as previously noted, other mass or molar ratios may beused.

In some examples, the resin precursor also includes an epoxy silane oranother reactive silane that can be incorporated into the cross-linkedPOSS resin matrix. The epoxy silane includes an epoxy group at one endof the molecule and a silane at the other end of the molecule. The epoxygroup can be incorporated covalently into the epoxy POSS resin film (byreaction of the epoxy group) and the silane group can covalently attachto surface groups (e.g., —OH) of the support 12. The epoxy silane may beincluded when the support 12 does not include surface-activatingagent(s) that can adhere the epoxy POSS resin film to the support 12.However, it is to be understood that epoxy silane may be excluded whenthe support 12 is a silica-based substrate with a suitablesurface-activating agent that can adhere the epoxy POSS resin film tothe support 12.

In other examples when the support 12 does not includesurface-activating agent(s), the resin precursor used to form thecross-linked epoxy POSS resin film 14 may not include the epoxy silaneor other reactive silane. Rather, the epoxy silane or other reactivesilane and at least one of the epoxy POSS monomeric unit(s) may bedeposited first to attach the silane to the support 12, and then thepreviously described resin precursor (without the silane) may be reactedwith the silane to form the cross-linked epoxy POSS resin film 14.

The resin precursor may also include a photoacid generator (PAG), asensitizer, a solvent, and/or a wetting agent. These components may beadded in any suitable amount to aid in polymerization and/or indeposition of the resin precursor. In some aspects of all of the methodsdescribed herein, the resin precursor comprises a wetting agent, such asa polyacrylate surfactant or a silicone surfactant.

In some examples, incompletely condensed silsesquioxane materials in thePOSS precursor (which contain silanol groups) react with the substratesurface to bind the resin to the surface.

Scheme 1 illustrates one example of the resin precursor and thecross-linked epoxy POSS resin film formed therefrom.

Scheme 1A depicts another example of the cross-linked resin film.

One of ordinary skill will understand that the polymers shown areexemplary, as individual monomers may combine in any order or polymericpattern, and additional monomers may be attached to the monomeric unitsshown, e.g., the pendant arms of the cages may be linked to furthermonomers.

As depicted, in this example, the resin precursor is formed by mixingthe epoxy silane (which can attach to the support 12 via an oxygenlinkage as illustrated in Scheme 1), epoxycyclohexylalkyl POSS, andglycidyl POSS in the presence of a sensitizer and PAG. The support-boundepoxy silane may in another example be of the structureSupport-O—Si(R)₂—O—C₂₋₆alkykepoxide), where each R is an alkyl groupsuch as a methyl or ethyl group. The resin precursor is formed incertain examples by mixing the support-bound epoxy resin with one or twodifferent epoxy POSS monomeric units. In other examples, the resinprecursor is deposited on the surface of the support 12 using anysuitable deposition method. Curing (i.e., polymerization andcross-linking) of the resin precursor is performed by exposure toactinic radiation (such as ultraviolet (UV) radiation). This processresults in the cross-linked epoxy POSS resin film 14. The ratio ofmonomers within the final cross-linked epoxy POSS resin film 14 dependsupon the stoichiometry of the monomers in the initial resin precursormixture.

In some examples of the method shown in FIGS. 1A through 1F, thecross-linked epoxy POSS resin film 14 may be exposed to a hard bakeafter curing. The hard bake helps to drive the cross-linking reaction tocompletion (e.g., UV initiates the polymerization/cross-linking processand the reaction continues in the dark until complete). The hard bakealso incubates or dehydrates the cross-linked epoxy POSS resin film 14to drive out any solvent(s) that may remain after curing. The durationof the hard bake may last from about 5 seconds to about 10 minutes at atemperature ranging from about 100° C. to about 300° C. An example of adevice that can be used for hard baking includes a hot plate.

As illustrated in FIG. 1A, in some examples, the cross-linked epoxy POSSresin film 14 is not imprinted.

As shown between FIGS. 1A and 1B, two different Routes A or B may beperformed. In Route A, the patterned hydrophobic polymer layer 16 isformed on the cross-linked epoxy POSS resin film 14 without furtherprocessing of the cross-linked epoxy POSS resin film 14. In Route B, thepatterned hydrophobic polymer layer 16 is formed on the cross-linkedepoxy POSS resin film 14 after additional processing is performed tointroduce functional groups to the cross-linked epoxy POSS resin film 14that can covalently bond to a functional group of a subsequently appliedpolymer coating 22.

With Route A, the cross-linked epoxy POSS resin film 14 is not exposedto additional processing before the patterned hydrophobic polymer layer16 is formed. As such, the patterned hydrophobic polymer layer 16 isformed on the as-formed cross-linked epoxy POSS resin film 14.

The patterned hydrophobic polymer layer 16 may be made up of any polymerthat is more hydrophobic than the cross-linked epoxy POSS resin film 14and that does not adhere to the subsequently deposited polymer coating22. Examples of the hydrophobic polymer include a fluorinated polymer, anegative tone photoresist, or a polysiloxane. The fluorinated polymermay be an amorphous (non-crystalline) fluoropolymer (e.g., CYTOP® fromBellex), a crystalline fluoropolymer, or a fluoropolymer having bothamorphous and crystalline domains. Any suitable negative tonephotoresist may be used, such as epoxy-based negative photoresists(e.g., the SU-8 series from MicroChem). Any suitable polysiloxane mayalso be used, such as polydimethylsiloxane (PDMS).

The patterned hydrophobic polymer layer 16 may be formed via anysuitable technique. In one example to form the patterned hydrophobicpolymer layer 16, the hydrophobic polymer is deposited (e.g., spincoated, etc.) on the cross-linked epoxy POSS resin film 14 and thedeposited hydrophobic polymer is patterned using nanoimprint lithographyand/or photolithography. In another example to form the patternedhydrophobic polymer layer 16, the hydrophobic polymer is deposited inthe desired pattern on the cross-linked epoxy POSS resin film 14 usinginkjet printing and/or microcontact printing.

The patterned hydrophobic polymer layer 16 may be a continuous layerwhich includes interstitial regions 20 separating adjacent depressions18. At each depression 18, discrete areas of the cross-linked epoxy POSSresin film 14 are exposed (as shown in FIG. 1B).

Many different layouts of the depressions 18 may be envisaged, includingregular, repeating, and non-regular patterns. In an example, thedepressions 18 are disposed in a hexagonal grid for close packing andimproved density. Other layouts may include, for example, rectilinear(i.e., rectangular) layouts, triangular layouts, and so forth. Thelayout or pattern can be an x-y format of depressions 18 that are inrows and columns. In some other examples, the layout or pattern can be arepeating arrangement of depressions 18 and/or interstitial regions 20.In still other examples, the layout or pattern can be a randomarrangement of depressions 18 and/or interstitial regions 20. Thepattern may include spots, pads, wells, posts, stripes, swirls, lines,triangles, rectangles (e.g., defining flow channels), circles, arcs,checks, plaids, diagonals, arrows, squares, and/or cross-hatches. Insome examples, the pattern includes wells. Still other examples ofpatterned surfaces that can be used in the examples set forth herein aredescribed in U.S. Pat. Nos. 8,778,849; 9,079,148; 8,778,848; and U.S.Patent Publication No. 2014/0243224, each of which is incorporatedherein by reference in its entirety.

The layout or pattern may be characterized with respect to the densityof the depressions 18 (i.e., number of depressions 18) in a definedarea. For example, the depressions 18 may be present at a density ofapproximately 2 million per mm². The density may be tuned to differentdensities including, for example, a density of at least about 100 permm², about 1,000 per mm², about 0.1 million per mm², about 1 million permm², about 2 million per mm², about 5 million per mm², about 10 millionper mm², about 50 million per mm², or more. Alternatively oradditionally, the density may be tuned to be no more than about 50million per mm², about 10 million per mm², about 5 million per mm²,about 2 million per mm², about 1 million per mm², about 0.1 million permm², about 1,000 per mm², about 100 per mm², or less. It is to befurther understood that the density of depressions 18 defined by thepatterned hydrophobic polymer layer 16 can be between one of the lowervalues and one of the upper values selected from the ranges above. Asexamples, a high density array may be characterized as havingdepressions 18 separated by less than about 1 μm of interstitial region20, and a low density array may be characterized as having depressions18 separated by greater than about 1 μm of interstitial region 20.

The layout or pattern may also or alternatively be characterized interms of the average pitch, i.e., the spacing from the center of thedepressions 18 to the center of an adjacent interstitial region 20(center-to-center spacing). The pattern can be regular such that thecoefficient of variation around the average pitch is small, or thepattern can be non-regular in which case the coefficient of variationcan be relatively large. In either case, the average pitch can be, forexample, at least about 10 nm, about 0.1 μm, about 0.5 μm, about 1 μm,about 5 μm, about 10 μm, about 100 μm, or more. Alternatively oradditionally, the average pitch can be, for example, at most about 100μm, about 10 μm, about 5 μm, about 1 μm, about 0.5 μm, about 0.1 μm, orless. The average pitch for a particular pattern of depressions 18 canbe between one of the lower values and one of the upper values selectedfrom the ranges above. In an example, the depressions 18 have a pitch(center-to-center spacing) of about 1.5 μm.

In the example shown in FIG. 1B, the depressions 18 are wells. The wellsmay be micro wells or nanowells. Each well may be characterized by itsvolume, well opening area, depth, and/or diameter.

Each well can have any volume that is capable of confining a liquid. Theminimum or maximum volume can be selected, for example, to accommodatethe throughput (e.g. multiplexity), resolution, analyte composition, oranalyte reactivity expected for downstream uses of the array 10′ (shownin FIG. 1F). For example, the volume can be at least about 1×10⁻³ μm³,about 1×10⁻² μm³, about 0.1 μm³, about 1 μm³, about 10 μm³, about 100μm³, or more. Alternatively or additionally, the volume can be at mostabout 1×10⁴ μm³, about 1×10³ μm³, about 100 μm³, about 10 μm³, about 1μm³, about 0.1 μm³, or less.

The area occupied by each well opening on a surface can be selectedbased upon similar criteria as those set forth above for well volume.For example, the area for each well opening on a surface can be at leastabout 1×10⁻³ μm², about 1×10⁻² μm², about 0.1 μm², about 1 μm², about 10μm², about 100 μm², or more. Alternatively or additionally, the area canbe at most about 1×10³ μm², about 100 μm², about 10 μm², about 1 μm²,about 0.1 μm², about 1×10⁻² μm², or less.

The depth of each well can be at least about 0.1 μm, about 1 μm, about10 μm, about 100 μm, or more. Alternatively or additionally, the depthcan be at most about 1×10³ μm, about 100 μm, about 10 about 1 μm, about0.1 μm, or less.

In some instances, the diameter of each well can be at least about 50nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 10 μm, about 100 μm,or more. Alternatively or additionally, the diameter can be at mostabout 1×10³ μm, about 100 μm, about 10 μm, about 1 μm, about 0.5 μm,about 0.1 μm, or less (e.g., about 50 nm).

With Route B shown between FIGS. 1A and 1B, the cross-linked epoxy POSSresin film 14 is exposed to additional processing before the patternedhydrophobic polymer layer 16 is formed thereon in the manner previouslydescribed.

This additional processing may include plasma ashing or a chemicaltreatment to introduce hydroxyl groups to the cross-linked epoxy POSSresin film 14. In some examples, the processing is oxygen plasma ashing,and the process introduces free —OH groups (e.g., hydroxyl and/orcarboxyl groups) to the resin film. Scheme 2 illustrates one example ofthe introduction of hydroxyl groups to the cross-linked epoxy POSS resinfilm 14.

Scheme 2A illustrates another example of the introduction of hydroxylgroups to the cross-linked epoxy POSS resin film 14.

The hydroxyl group containing cross-linked epoxy POSS resin film 14 maythen exposed to silanization or another chemical process to introducefunctional groups (e.g., “FG” in FIG. 1E) that can attach to thehydroxyl group(s). These functional groups FG may be anchor moleculesthat enhance the attachment of the subsequently applied polymer coating22 to the cross-linked epoxy POSS resin film 14 exposed in thedepressions 18. As such, the selection of the functional group FG maydepend, in part, upon the molecule that is to be used to form thepolymer coating 22 (shown in FIG. 1C), as it may be desirable to form acovalent bond and/or a non-covalent bond (e.g., van der Waals orHydrogen) between the functional group FG and the subsequently depositedpolymer coating 22. Examples of the functional groups FG are selectedfrom the group consisting of:

wherein n ranges from 1 to 20,

and wherein - - - represents an alkylsilane (e.g., by reaction of thehydroxyl groups with a trialkoxyalkylsilane), a poly(ethyleneglycol)-silane (e.g., by reaction of the hydroxyl groups with atrialkoxysilane poly(ethylene glycol)), or an alkyl halide-silane (e.g.,by reaction of the hydroxyl groups with an alkyl halide), or apolyethylene glycol chain, or any other silane that can form a tripodalconnection with the hydroxyl group(s), or another group that can form aC—C—O— connection at the hydroxyl group(s). These functional groups, orany other functional groups that can withstand the processing that isperformed during the formation of the patterned hydrophobic coatinglayer 16, may be used. The process conditions may also be adjusted toutilize a desirable functional group. While several examples offunctional groups have been provided, it is to be understood that otherhydrophilic or hydrophobic functional groups that can be covalentlybonded to, or entrapped by the epoxy POSS resin film 14, and thatintroduces a desirable functionality to the epoxy POSS resin film 14 maybe used. Still further, derivatives of the various functional groups FGand/or substituted variations of the functional groups FG may be used.In other examples, the polymer coating can be coated and cured to thecross-linked epoxy POSS resin film directly after plasma ashing, withouta separate silanization or functionalization step.

The method used to attach the functional group FG to the hydroxyl groupsof the cross-linked epoxy POSS resin film 14 may vary depending upon thefunctional group FG that is being used. Examples of suitable methodsinclude vapor deposition, the YES method, solution deposition methods,or other deposition methods.

With Route B, the cross-linked epoxy POSS resin film 14 is modified toform a functionalized cross-linked epoxy POSS resin film 14′. Thepatterned hydrophobic polymer layer 16 (including its interstitialregions 20 and depressions 18) may then be formed in the mannerpreviously described on the functionalized cross-linked epoxy POSS resinfilm 14′. In this example, discrete portions of the functionalizedcross-linked epoxy POSS resin film 14′ are exposed at the depressions18.

Whether Route A or Route B is performed, after the patterned hydrophobicpolymer layer 16 is formed, the polymer coating 22 is applied or grownon the patterned hydrophobic polymer layer 16 and in the depressions 18.This is shown in FIG. 1C.

The polymer coating 22 may be deposited on the patterned hydrophobicpolymer layer 16 and on the exposed surfaces of the cross-linked epoxyPOSS resin film 14 or the functionalized cross-linked epoxy POSS resinfilm 14′ using spin coating, dipping or dip coating, spray coating, orthe like. In an example, the polymer coating 22 is deposited as asolution, an example of which includes PAZAM in an ethanol and watermixture. Any solvent or solvent combination may be used that aids inwetting. Surfactants may also be added to the solution to aid inwetting.

After being coated, the polymer coating 22 may be exposed to a curingprocess to form attached coating portion(s) 22′ (where the polymercoating 22 attaches to the exposed cross-linked epoxy POSS resin film 14or functionalized cross-linked epoxy POSS resin film 14′ in thedepressions 18) and unattached coating portion(s) 22″ (where polymercoating 22 does not attach to the patterned hydrophobic polymer layer 16(e.g., at the interstitial regions 20)). The curing temperature mayrange from about 20° C. to about 80° C. and the curing time may rangefrom seconds to about 120 minutes. In an example, curing the polymercoating 22 may take place at about 60° C. for about 1 hour. Curingtemperature and time may vary depending, in part, on the polymer coating22 being formed.

When Route B is utilized to form the functionalized cross-linked epoxyPOSS resin film 14′, the polymer coating 22 may be grown from thesurface of the resin film 14′. For example, the support 12 having theresin film 14′ and the patterned hydrophobic polymer layer 16 may beimmersed into a suitable bath containing monomer(s) and an initiator.Polymerization of the monomer(s) will form the attached portion(s) 22′of the polymer coating 22.

The attached and unattached coating portion(s) 22′, 22″ are shown inFIG. 1C. The mechanism for attachment of the attached coating portion(s)22′ will depend upon whether the cross-linked epoxy POSS resin film 14is present (Route A) or whether the functionalized cross-linked epoxyPOSS resin film 14′ is present (Route B).

As an example, the polymer coating 22 can attach to, or be inserted intounreacted epoxy groups of the cross-linked epoxy POSS resin film 14 toform the attached coating portion(s) 22′. For example, free amines onthe polymer structure (e.g., Formula (I)) may react with unreacted epoxygroups in the cross-linked epoxy POSS resin film 14.

As another example, the polymer coating 22 can attach to the addedfunctional group(s) FG of the functionalized cross-linked epoxy POSSresin film 14′ to form the attached coating portion(s) 22′. The reactionthat takes place will depend upon the functional group FG of thefunctionalized cross-linked epoxy POSS resin film 14′ and the functionalgroup of the polymer coating 22. The following are some examples of thereactions that can take place.

When the functional group FG of the functionalized cross-linked epoxyPOSS resin film 14′ is norbornene or a norbornene derivative, thenorbornene or a norbornene derivative can: i) undergo a 1,3-dipolarcycloaddition reaction (i.e., click reaction) with an azide/azido groupof PAZAM or a Formula (I) polymer; ii) undergo a coupling reaction witha tetrazine group attached to the polymer structure (e.g., Formula (I));iii) undergo a cycloaddition reaction with a hydrazone group attached tothe polymer structure (e.g., Formula (I)); iv) undergo a photo-clickreaction with a tetrazole group attached to the polymer structure (e.g.,Formula (I)); or v) undergo a cycloaddition with a nitrile oxide groupattached to the polymer structure (e.g., Formula (I)). An example of thenorbornene or a norbornene functional group undergoing the 1,3-dipolarcycloaddition reaction with the azide/azido group of PAZAM is shown inScheme 3.

where the —CH₂C(O)NHR group is the side chain of the PAZAM polymer.

In other examples, the functional group FG of the functionalizedcross-linked epoxy POSS resin is introduced at the hydroxyl positionsthat were added by the surface functionalization methods describedabove. An example is shown in Scheme 3A, where FG is a functional groupas described herein.

In some examples, the added functional groups comprise alkene orcycloalkane groups. In an example, such groups are shown in Scheme 3B.

In an example, introduction of the polymer coating 22 is accomplished byreaction of the polymer material, such as a polymer of Formula (I), orPAZAM, or a combination of SFA and azido- or bromo-functionalized SFA,with the appended functional groups. An example is shown in Scheme 3C,showing just one reaction site on the POSS resin film. One of ordinaryskill will recognize that reaction of the polymer coating with thefunctionalized POSS resin film occurs at multiple locations of thepolymer and resin.

When the functional group FG of the functionalized cross-linked epoxyPOSS resin film 14′ is cyclooctyne or a cyclooctyne derivative, thecyclooctyne or cyclooctyne derivative can: i) undergo a strain-promotedazide-alkyne 1,3-cycloaddition (SPAAC) reaction with an azide/azido ofPAZAM (or other polymer such as a Formula (I) polymer), or ii) undergo astrain-promoted alkyne-nitrile oxide cycloaddition reaction with anitrile oxide group attached to a polymer (such as Formula (I)).

When the functional group FG of the functionalized cross-linked epoxyPOSS resin film 14′ is a bicyclononyne, the bicyclononyne can undergosimilar SPAAC alkyne cycloaddition with azides or nitrile oxidesattached to PAZAM (or other suitable polymer material such as a Formula(I) polymer) due to the strain in the bicyclic ring system.

After the attached and unattached coating portion(s) 22′, 22″ areformed, the unattached coating portion(s) 22″ may be washed off of thepatterned hydrophobic layer 16 (and in some instances off of theattached coating portion(s) 22′). The washing process may utilize awater bath and sonication. The water bath may be maintained at arelatively low temperature ranging from about 20° C. to about 60° C.FIG. 1D shows the array 10 after the unattached coating portion(s) 22″is/are removed.

FIG. 1E is an enlarged view of one of the depressions 18 after theattached coating portion 22′ has been formed therein. In the exampleshown in FIG. 1E, the functionalized cross-linked epoxy POSS resin film14′ is formed and norbornene silane is the functional group FG that isadded to the surface of the cross-linked epoxy POSS resin film 14′. ThePAZAM attaches to the functional group FG to form the attached coatingportion 22′ within the depression 18 defined by the patternedhydrophobic polymer layer 16.

Referring now to FIG. 1F, an amplification primer 24 may be grafted tothe attached polymer coating portion 22′. Examples of suitable primers24 include forward amplification primers or reverse amplificationprimers. Specific examples of suitable primers 24 include P5 or P7primers, which are used on the surface of commercial flow cells sold byIllumina Inc. for sequencing on HiSeq®, HiSeqX®, MiSeq®, NextSeq® andGenome Analyzer® instrument platforms.

The amplification primer 24 may be modified at the 5′ end with a groupthat is capable of reacting with a functional group of the attachedcoating portion 22′ (e.g., the azide shown in FIG. 1E). For example, abicyclo[6.1.0] non-4-yne (BCN) terminated primer may be captured by anazide of the attached coating portion 22′ via strain-promoted catalystfree click chemistry. For another example, an alkyne terminated primermay be captured by an azide of the attached coating portion 22′ viacopper catalyzed click chemistry. For still another example, anorbornene terminated primer, may be undergo a catalyst-free ring strainpromoted click reaction with a tetrazine functionalized attached coatingportion 22′. Other examples of terminated primers that may be usedinclude a tetrazine terminated primer, an azido terminated primer, anamino terminated primer, an epoxy or glycidyl terminated primer, athiophosphate terminated primer, a thiol terminated primer, an aldehydeterminated primer, a hydrazine terminated primer, and a triazolinedioneterminated primer. Other examples of terminated primers arethiophosphate-terminated primers.

Grafting may be accomplished by dunk coating, spray coating, puddledispensing, or by another suitable method that will attach the primer(s)24 to attached coating portions 22′ in at least some of the depressions18. Each of these examples may utilize a primer solution or mixture,which may include the primer(s) 24, water, a buffer, and an optionalcatalyst(s).

Dunk coating may involve submerging (via an automated or manual process)the array 10 (shown in FIG. 1D) into a series of temperature controlledbaths. The baths may include the primer solution or mixture. Throughoutthe various baths, the primer(s) 24 will attach to the attached coatingportions 22′ in at least some of the depression(s) 18. In an example,the array 10 will be introduced into a first bath including the primersolution or mixture where a reaction takes place to attach the primer(s)24, and then the array 10′ will be moved to additional baths forwashing.

Spray coating may be accomplished by spraying the primer solution ormixture directly onto the array 10. The spray coated array may beincubated for a time ranging from about 5 minutes to about 60 minutes ata temperature ranging from about 10° C. to about 70° C. Afterincubation, the primer solution or mixture may be diluted and removedusing, for example, a spin coater.

Puddle dispensing may be performed according to a pool and spin offmethod, and thus may be accomplished with a spin coater. The primersolution or mixture may be applied (manually or via an automatedprocess) to the array 10. The applied primer solution or mixture may beapplied to or spread across the entire surface of the array 10. Theprimer coated array 10 may be incubated for a time ranging from about 5minutes to about 60 minutes at a temperature ranging from about 10° C.to about 80° C. After incubation, the primer solution or mixture may bediluted and removed using, for example, the spin coater.

After grafting, the desired surface chemistry has been applied, and thearray 10′ may be used in a variety of sequencing approaches ortechnologies.

The example of the method shown in FIGS. 1A-1F may also be performedwith modified epoxy POSS monomeric unit. In this example, the resinprecursor includes the epoxy POSS monomeric unit(s) previously describedand a modified epoxy POSS monomeric unit. In these examples, at leastone of R₁ through R₈ or R₁₀ or R₁₂ of the modified epoxy POSS monomericunit is an epoxy group (for incorporation into the epoxy POSS resin film14) and at least one of R₁ through R₈ or R₁₀ or R₁₂ is anotherfunctional group that can covalently or non-covalently bond to afunctional group of a subsequently applied polymer coating 22. As such,in this example, the other functional group is incorporated directlyinto the POSS core or cage structure. Examples of the other functionalgroup include any of the examples of the functional group FG.

Resin precursors including the modified epoxy POSS monomeric unit(s) mayinclude from about 50 mol % to about 90 mol % of the epoxy POSSmonomeric unit(s) and from about 10 mol % to about 50 mol % of themodified epoxy POSS monomeric unit(s) (i.e., 100 mol %-X mol % of theepoxy POSS monomeric unit(s)). As such, the mass or molar ratio of epoxyPOSS monomeric unit(s) to modified epoxy POSS monomeric unit(s) in someexamples of the resin precursors ranges from about 1:1 to about 9:1. Inthese resin precursors, when two different (non-modified) epoxy POSSmonomeric units are used in combination, any suitable mass or molarratio of the two units may be selected. For example, a first epoxy POSSmonomeric unit (e.g., epoxycyclohexylalkyl POSS) may be present in anamount (Y) ranging from about 10 mol % to about 90 mol % of the totalamount of epoxy POSS monomeric units and a second epoxy POSS monomericunit (e.g., glycidyl POSS) may make up the balance of the total epoxyPOSS monomeric units (i.e., 100 mol % of epoxy POSS monomeric units-Ymol %). In other examples, any of the epoxy POSS monomeric units and anyof the modified epoxy POSS monomeric units may be present in an amountranging from about 10 mol % to about 90 mol %.

The use of the modified epoxy POSS monomeric unit to form the resin filmintroduces the functional group FG directly into the backbone of theresin film, and thus provides a site (other than the unreacted epoxygroups) for attachment of the polymeric coating 22 without having toperform further processes on the resin film as described in Route B.

FIGS. 2A through 2D together illustrate another example of the methoddisclosed herein, which forms another example of the array disclosedherein. FIG. 2C is an enlarged view of a depression of the array that isformed.

In the example method(s) shown in FIGS. 2A through 2D, a modified epoxyPOSS resin film 14″ is formed, which includes a controlled radicalpolymerization (CRP) agent (shown schematically as 26 in FIG. 2C)incorporated into the cross-linked matrix. The CRP agent 26 may be areversible addition-fragmentation chain transfer (RAFT) agent or an atomtransfer radical polymerization (ATRP) initiator. For the RAFT agents,the orientation of the thiocarbonyl group at the surface affects thepolymerization. In one example, the RAFT agent is capable of covalentlyattaching to the surface of the modified epoxy POSS resin film 14″ viathe stabilizing group such that the growing radical chain moves awayfrom the surface. This is referred to as the Z-group approach. Inanother example, the RAFT attaches to the surface via a leaving andinitiating group (i.e., the R-group approach). The R-group approach mayafford greater control over the molecular weight, and chain-chaincoupling may be minimized.

As will be described further herein, the CRP agent 26 may beincorporated into the cross-linked matrix during curing or after curing,and may be incorporated into the backbone of the cross-linked matrix(via a non-POSS monomeric unit or a modified epoxy POSS monomeric unit)or may be attached to the backbone via another functional group.

FIG. 2A illustrates a support 12 with the modified epoxy POSS resin film14″ formed thereon. Any example of the support 12 previously describedherein may be used. In an example, the modified epoxy POSS resin film14″ is formed on the support 12, and FIG. 2A illustrates three routes,shown as Route C, Route D, and Route E, for forming the modified epoxyPOSS resin film 14″.

Using Route C, a resin precursor is formed which includes aCRP-containing monomeric unit, the resin precursor is deposited on asurface of the support 12, and the resin precursor is irradiated with UVlight to cure and form the cross-linked epoxy POSS resin film 14″. Assuch, Route C involves incorporating the CRP agent 26 into the backboneof the cross-linked matrix of the resin film 14″ during curing of theresin precursor.

In this example, the resin precursor is a mixture, which includes atleast an epoxy POSS monomeric unit and a CRP-containing monomeric unit.Any examples of the epoxy POSS monomeric units described herein may beused. The CRP-containing monomeric unit may be a non-POSS monomeric unitor a modified epoxy POSS monomeric unit.

The CRP-containing non-POSS monomeric unit does not include a POSS core.Rather, the CRP agent 26 is tethered to a functional group that can beincorporated covalently into the modified epoxy POSS resin film 14″ withthe epoxy POSS monomeric unit(s). As an example, the CRP agent 26 may bereacted with an epoxy functional group to form an epoxy-functionalizedCRP agent, such as an epoxy-functionalized RAFT agent (Scheme 4) or anepoxy-functionalized ATRP initiator (Scheme 5).

In Scheme 4, the RAFT agent is2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid3-azido-1-propanol ester and the epoxy functional group is glycidylpropargyl ether. It is to be understood that other commerciallyavailable RAFT agents or other prepared RAFT agents may be used. Anothersuitable RAFT agent is4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol:

which can be reacted with any epoxyhalohydrin (e.g., epichlorohydrin

to form another example of the epoxy-functionalized RAFT agent.

In Scheme 5, the ATRP initiator is 2-azidoethyl 2-bromoisobutyrate andthe epoxy functional group is glycidyl propargyl ether. It is to beunderstood that other commercially available ATRP initiators or otherprepared ATRP initiators may be used, such as Poly(ethylene glycol)methyl ether 2-bromoisobutyrate.

As mentioned above, the CRP-containing monomeric unit may be a modifiedepoxy POSS monomeric unit. In these examples, the monomeric unit is thePOSS core having an epoxy group (for incorporation into the epoxy POSSresin film 14″) as at least one of R₁ through R₈ or R₁₀ or R₁₂ andhaving the CRP agent 26 as at least one of R₁ through R₈ or R₁₀ or R₁₂.As such, in this example, the CRP agent 26 is incorporated directly intothe POSS core or cage structure. As examples, any RAFT agent with thestructure

and any ATRP initiator with the structure

may be used, e.g., 2-azidoethyl 2-bromoisobutyrate, 2-bromoisobutyricanhydride, bromoisobutyryl bromide, or poly(ethylene glycol)bis(2-bromoisobutyrate). For example, the RAFT agent may be:

Other suitable RAFT agents are dithiobenzoates, trithiocarbonates, anddithiocarbamates. The specific RAFT agents or ATRP initiators previouslymentioned may also be used to modify the epoxy POSS core/cage.

In Route C, the resin precursor including the CRP-containing monomericunit(s) may include from about 50 mol % to about 90 mol % of the epoxyPOSS monomeric unit(s) and from about 10 mol % to about 50 mol % of theCRP-containing monomeric unit(s) (i.e., 100 mol %-X mol % of the epoxyPOSS monomeric unit(s)). As such, the molar or mass ratio of epoxy POSSmonomeric unit(s) to CRP-containing monomeric unit(s) in some examplesof the resin precursors ranges from about 1:1 to about 9:1. As oneexample, the molar or mass ratio of total epoxy POSS monomeric units(e.g., epoxycyclohexylalkyl POSS and glycidyl POSS) toepoxy-functionalized CRP agent(s) ranges from about 1:1 to about 9:1. Inthese resin precursors, when two different (non-modified) epoxy POSSmonomeric units are used in combination, any suitable mass or molarratio of the two units may be selected. For example, a first epoxy POSSmonomeric unit (e.g., epoxycyclohexylalkyl POSS) may be present in anamount (Y) ranging from about 10 mol % to about 90 mol % of the totalamount of epoxy POSS monomeric units and a second epoxy POSS monomericunit (e.g., glycidyl POSS) may make up the balance of the total epoxyPOSS monomeric units (i.e., 100 mol % of epoxy POSS monomeric units-Ymol %). In other examples, any of the epoxy POSS monomeric units and anyof the CRP-containing monomeric unit(s) may be present in an amountranging from about 10 mol % to about 90 mol %.

In some examples using Route C, the resin precursor also includes anepoxy silane or another reactive silane that can be incorporated intothe cross-linked POSS resin matrix. The epoxy silane includes an epoxygroup at one end of the molecule and a silane at the other end of themolecule. The epoxy group can be incorporated covalently into themodified epoxy POSS resin film 14″ and the silane group can covalentlyattach to surface groups (e.g., —OH) of the support 12. The epoxy silanemay be included when the support 12 does not include surface-activatingagent(s) that can adhere the epoxy POSS resin film 14″ to the support12. However, it is to be understood that epoxy silane may be excludedwhen the support 12 is a silica-based substrate with a suitablesurface-activating agent that can adhere the epoxy POSS resin film 14″to the support 12.

In other examples when the support 12 does not includesurface-activating agent(s), the resin precursor used to form themodified cross-linked epoxy POSS resin film 14″ may not include theepoxy silane or other reactive silane. Rather, the epoxy silane or otherreactive silane and at least one of the epoxy POSS monomeric unit(s) maybe deposited first to attach the silane to the support 12, and then thepreviously described resin precursor for Route C (without the silane)may be reacted with the silane to form the modified epoxy POSS resinfilm 14″.

The resin precursor used in Route C may also include a photoacidgenerator (PAG), a sensitizer, a solvent, and/or a wetting agent. Thesecomponents may be added in any suitable amount to aid in polymerizationand/or in deposition of the resin precursor.

Schemes 6 and 7 illustrate examples of the resin precursors used inRoute C and the modified epoxy POSS resin film formed therefrom. Theseexamples illustrate the use of CRP-containing non-POSS monomeric unit.

One of ordinary skill will recognize the POSS resin films depicted inSchemes 6 and 7 may also be depicted as shown in the preceding Schemes,and that the resin films are composed of monomeric units in any order orpolymeric pattern (e.g., random, block, alternating, or combinationsthereof).

In these examples, the resin precursor is formed by mixing theCRP-containing non-POSS monomeric unit (i.e., the epoxy-functionalizedRAFT agent or the epoxy-functionalized ATRP initiator), epoxy silane orother reactive silane (which can attach to the support 12 via an oxygenlinkage as illustrated in Schemes 6 and 7), epoxycyclohexylalkyl POSS,and glycidyl POSS in the presence of a sensitizer and PAG. The resinprecursor is deposited on the surface of the support 12 using anysuitable deposition method. Curing (i.e., polymerization andcross-linking) of the resin precursor is performed by exposure toactinic radiation (such as ultraviolet (UV) radiation). This processresults in the cross-linked epoxy POSS resin films 14″. The ratio ofmonomers within the final cross-linked epoxy POSS resin film 14″ dependsupon the stoichiometry of the monomers in the initial resin precursormixture.

As illustrated in both Schemes 6 and 7, the CRP-containing non-POSSmonomeric unit introduces a polymer growth initiation site (i.e., CRPinitiation site) into the backbone of the cross-linked matrix of theresin film 14″ during curing of the resin precursor. While not shown, itis to be understood that when the CRP-containing epoxy POSS monomericunit is used instead of the CRP-containing non-POSS monomeric unit, thebackbone of the cross-linked matrix will include an additional POSS cageto which the polymer growth initiation site (i.e., CRP initiation site)is attached.

Route D involves attaching the CRP agent 26 to the backbone of thecross-linked matrix via another functional group after the resin film14″ has been cured.

In this example, the resin precursor is a mixture similar to thatdescribed in reference to FIG. 1A. For example, the resin precursor inRoute D may include the epoxy POSS monomeric unit(s), the epoxy silaneor other reactive silane (e.g., when attachment to the support 12 isdesirable), the photoacid generator (PAG), the sensitizer, the solvent,and/or the wetting agent. The resin precursor is deposited on thesurface of the support 12 using any suitable deposition method. Curing(i.e., polymerization and cross-linking) of the resin precursor isperformed by exposure to actinic radiation (such as ultraviolet (UV)radiation). This process results in a cross-linked epoxy POSS resinfilm, similar to resin film 14 previously described (see, e.g., Scheme1). A hard bake may be performed as previously described. In Route D, itis to be understood that the epoxy silane or other reactive silane andat least one of the epoxy POSS monomeric unit(s) may be deposited firstto attach the silane to the support 12, and then the previouslydescribed resin precursor for Route D (without the silane) may bereacted with the silane to form the cross-linked epoxy POSS resin film14.

The cross-linked epoxy POSS resin film is then exposed to plasma ashingor a chemical treatment to introduce —OH groups (e.g., hydroxyl (C—OH orSi—OH) and/or carboxyl groups) to the cross-linked epoxy POSS resin film(e.g., as shown in Scheme 2).

The hydroxyl group containing cross-linked epoxy POSS resin film maythen be exposed to silanization or another chemical process to introducea functional group at the hydroxyl group, where the selected functionalgroup can attach to a desired CRP agent 26. As such, the selection ofthe functional group in Route D may depend, in part, upon the CRP agent26 that is to be attached. For example, a RAFT agent with a terminalazide group can react with an alkyne functional group that has beenattached at the hydroxyl group of the cross-linked epoxy POSS resinfilm.

Examples of suitable functional groups for Route D are selected from thegroup consisting of

wherein n ranges from 1 to 20,

and wherein - - - represents a species that is capable of reacting withthe —OH group of the cross-linked epoxy POSS resin film. Examplesof - - - include an alkylsilane (e.g., by reaction of the hydroxylgroups with a trialkoxyalkylsilane), a poly(ethylene glycol)-silane(e.g., by reaction of the hydroxyl groups with a trialkoxysilanepoly(ethylene glycol)), or an alkyl halide-silane (e.g., by reaction ofthe hydroxyl groups with an alkyl halide), or a polyethylene glycolchain, or any other silane that can form a tripodal connection with thehydroxyl group(s), or another group that can form a C—C—O— connection atthe hydroxyl group(s). Some specific examples include of the functionalgroups include silane PEG azide (Polysciences, Inc.), silane PEG alkyne(Polysciences, Inc.), 3-azidopropyltriethoxysilane (Gelest), or(Bicyclo[2.2.1]hept-5-en-2-yl)triethoxysilane. While several exampleshave been provided, it is to be understood that any functional groupthat can attach to the hydroxyl group(s) of the cross-linked epoxy POSSresin film and to the CRP agent 26 may be used.

The method used to attach the functional group to the hydroxyl groups ofthe cross-linked epoxy POSS resin film may vary depending upon thefunctional group that is being used. Examples of suitable methodsinclude vapor deposition, the YES method, solution deposition methods(e.g., dunk coating), or other deposition methods.

Any of the CRP agents 26 previously described (e.g.,2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid3-azido-1-propanol ester,4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol, etc.) maythen be attached to the functional group of the cross-linked epoxy POSSresin film. The method used to attach the CRP agents 26 to thefunctional group of the cross-linked epoxy POSS resin film may varydepending upon the CRP agent 26 that is being used. Examples of suitablemethods include solution deposition methods.

With Route D, the cross-linked epoxy POSS resin film is first modifiedto add the previously described functional group(s), and then the CRPagent 26 is attached to at least some of the functional groups to forman example of the modified epoxy POSS resin film 14″ (which includes apolymer growth initiation site).

Like Route D, Route E involves attaching the CRP agent 26 to thebackbone of the cross-linked matrix after the resin film 14″ has beencured.

In Route E, the resin precursor is a mixture including the epoxy POSSmonomeric unit(s) and a modified epoxy POSS monomeric unit. In thisexample of the modified epoxy POSS monomeric unit, at least one of R₁through R₈ or R₁₀ or R₁₂ is an epoxy group (for incorporation into theepoxy POSS resin film 14″) and at least one of R₁ through R₈ or R₁₀ orR₁₂ is a non-epoxy functional group that can attach to the CRP agent 26(e.g., the non-epoxy functional group may be selected from the groupconsisting of an azide, a thiol, a poly(ethylene glycol), a norbornene,and a tetrazine). This non-epoxy functional group will be integratedinto an initially modified epoxy POSS resin film during curing.

Resin precursors for Route E may include from about 50 mol % to about 90mol % of the epoxy POSS monomeric unit(s) and from about 10 mol % toabout 50 mol % of the modified epoxy POSS monomeric unit(s) (i.e., 100mol %-X mol % of the epoxy POSS monomeric unit(s)). As such, the molaror mass ratio of epoxy POSS monomeric unit(s) to modified epoxy POSSmonomeric unit(s) in some examples of the resin precursors ranges fromabout 1:1 to about 9:1. In these resin precursors, when two different(non-modified) epoxy POSS monomeric units are used in combination, anysuitable mass or molar ratio of the two units may be selected. Forexample, a first epoxy POSS monomeric unit (e.g., epoxycyclohexylalkylPOSS) may be present in an amount (Y) ranging from about 10 mol % toabout 90 mol % of the total amount of epoxy POSS monomeric units and asecond epoxy POSS monomeric unit (e.g., glycidyl POSS) may make up thebalance of the total epoxy POSS monomeric units (i.e., 100 mol % ofepoxy POSS monomeric units-Y mol %). In other examples, any of the epoxyPOSS monomeric units and any of the modified monomeric unit(s) may bepresent in an amount ranging from about 10 mol % to about 90 mol %.

The resin precursor for Route E may also include the epoxy silane orother reactive silane (e.g., when attachment to the support 12 isdesirable), the photoacid generator (PAG), the sensitizer, the solvent,and/or the wetting agent. The resin precursor is deposited on thesurface of the support 12 using any suitable deposition method. Curing(i.e., polymerization and cross-linking) of the resin precursor isperformed by exposure to actinic radiation (such as ultraviolet (UV)radiation). This process results in an initially modified epoxy POSSresin film, which includes the non-epoxy functional group. A hard bakemay be performed as previously described. In Route E, it is to beunderstood that the epoxy silane or other reactive silane and at leastone of the epoxy POSS monomeric unit(s) may be deposited first to attachthe silane to the support 12, and then the previously described resinprecursor for Route E (without the silane) may be reacted with thesilane to form the cross-linked epoxy POSS resin film 14″.

A desired CRP agent 26 may then be introduced to the initially modifiedepoxy POSS resin film to form the modified epoxy resin film 14″including the polymer growth initiation site. The CRP agent 26 may beattached to the non-epoxy functional group using any of the solutiondeposition techniques (e.g., dunk coating, etc.) previously describedherein. The CRP agent 26 will be selected so that it can react with anyof the non-epoxy functional groups disclosed herein (e.g., azide, thiol,poly(ethylene glycol), norbornene, or tetrazine functional groups).

Routes C, D, and E all result in the formation of the modified epoxyresin film 14″ on the support 12. The modified epoxy resin film 14″includes the polymer growth initiation site because of theattached/integrated CRP agent 26. As illustrated in FIG. 2A, themodified epoxy POSS resin film 14″ is not imprinted.

The patterned hydrophobic polymer layer 16 (including its interstitialregions 20 and depressions 18) may then be formed in the mannerpreviously described on the modified epoxy POSS resin film 14″. In thisexample, discrete portions of the modified epoxy POSS resin film 14″ areexposed at the depressions 18. The formed patterned hydrophobic polymerlayer 16 is shown in FIG. 2B.

The method of FIGS. 2A through 2D then involves growing the polymerbrush 28 from the polymer growth initiation site/CRP agent 26. The grownpolymer brush 28 is shown in FIG. 2C. Polymer growth may be performed indunk tanks which include the support 12 having the layers 14″ and 16thereon and a suitable monomer that is to be polymerized. Examples ofsuitable monomers include acrylamides (e.g., a PAZAM monomer or anotheracrylamide that is capable of attaching the primer 24), or acrylates.

Scheme 8 illustrates an example of the polymer brush formation. Thisscheme shows both an acrylamide (left) and an acrylate (right)polymerized from an ATRP initiator/CRP agent 26 (i.e.,

It is noted that the modified epoxy POSS resin film 14″ is representedby the silane group and the - - - between the silane group and CRP agent26. Polymer growth from acrylamide and acrylate monomers may also beaccomplished when the modified epoxy POSS resin film 14″ has a RAFT CRPagent 26 attached thereto.

The polymerization conditions may depend upon the monomer(s) and the CRPagent 26 of the modified epoxy POSS resin film 14″. As an example,solution-state conditions may be used for polymer brush growth.

FIG. 2B shows the array 10″ after the polymer brush 28 is formed in thedepressions 18 (exposed discrete areas of the modified epoxy POSS resinfilm 14″).

Referring now to FIG. 2D, an amplification primer 24 may be grafted tothe polymer brush 28. Any suitable amplification primer 24 may be used,and the amplification primer 24 may be modified at the 5′ end with agroup that is capable of reacting with a functional group of the polymerbrush 28. For example, a bicyclo[6.1.0] non-4-yne (BCN) terminatedprimer may be captured by an azide of the polymer brush 28 viastrain-promoted catalyst free click chemistry. For another example, analkyne terminated primer may be captured by an azide of the polymerbrush 28 via copper catalyzed click chemistry. For still anotherexample, a norbornene terminated primer, may be undergo a catalyst-freering strain promoted click reaction with a tetrazine functionalizedattached polymer brush 28. Other examples of terminated primers that maybe used include a tetrazine terminated primer, an azido terminatedprimer, an amino terminated primer, an epoxy or glycidyl terminatedprimer, a thiophosphate terminated primer, a thiol terminated primer, analdehyde terminated primer, a hydrazine terminated primer, and atriazolinedione terminated primer. Other terminated primers includethiophosphate-terminated primers.

Grafting may be accomplished as previously described, e.g., by dunkcoating, spray coating, puddle dispensing, or by another suitable methodthat will attach the primer(s) 24 to the polymer brush 28 in at leastsome of the depressions 18.

After grafting, the desired surface chemistry has been applied, and thearray 10′″ (FIG. 2D) may be used in a variety of sequencing approachesor technologies.

While several examples of the epoxy POSS resin film 14, 14′, 14″ havebeen disclosed herein, it is to be understood that layered epoxy POSSresin film 14, 14′, 14″ may be utilized, or that different epoxy POSSresin films 14, 14′, 14″ may be applied to/formed on different areas ofthe support 12. In the layered versions, different layers may be exposedat different areas in order to alter the functionality of the array atdifferent locations.

The arrays 10′, 10′″ disclosed herein may be used in a variety ofsequencing approaches or technologies, including techniques oftenreferred to as sequencing-by-synthesis (SBS), sequencing-by-ligation,pyrosequencing, and so forth. With any of these techniques, since theattached coating portion 22′ or polymer brush 28 and attached sequencingprimers 24 are present in the depressions 18 and not on the interstitialregions 20, amplification will be confined to the various depressions18.

Briefly, a sequencing by synthesis (SBS) reaction may be run on a systemsuch as the HiSeq®, HiSeqX®, MiSeq® or NextSeq® sequencer systems fromIllumina (San Diego, Calif.). A set of target DNA molecules to besequenced is hybridized to the bound amplification primers 24 and thenamplified, for example by kinetic exclusion amplification or by bridgeamplification. Denaturation leaves single-stranded templates anchored tothe attached coating portion 22′ or polymer brush 28, and severalmillion dense clusters of double-stranded DNA are generated (i.e.,cluster generation). The sequencing reactions are carried out.

The arrays 10′, 10′ disclosed herein may also be disposed in or formedas a part of a flow cell, which is a chamber including a solid surfaceacross which various carrier fluids, reagents, and so forth may beflowed. In an example, the flow cell may include the array 10′, 10′bonded to a top substrate through a sealing material (e.g., blackpolyimide or another suitable bonding material). The bonding may takeplace in bonding regions of the patterned hydrophobic polymer layer 16,the sealing material, and the top substrate. The bonding regions may belocated between flow channels so that the sealing material physicallyseparates one flow channel from an adjacent flow channel (to preventcross-contamination) and may be located at the periphery of the flowcell (to seal the flow cell from external contamination). It is to beunderstood, however, that the bonding regions and the sealing materialmay be located in any desired region depending on the implementation.Bonding may be accomplished via laser bonding, diffusion bonding, anodicbonding, eutectic bonding, plasma activation bonding, glass fritbonding, or others methods known in the art.

Other examples of flow cells and related fluidic systems and detectionplatforms that can be integrated with the array 10′, 10″ and/or readilyused in the methods of the present disclosure are described, forexample, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497; U.S.Pat. No. 7,057,026; WO 91/06678; WO 07/123744; U.S. Pat. Nos. 7,329,492;7,211,414; 7,315,019; 7,405,281, and US 2008/0108082, each of which isincorporated herein by reference in its entirety.

In some applications, the flow cell is used to perform controlledchemical or biochemical reactions in a reaction automation device, suchas in a nucleotide sequencer. Ports (not shown) may be drilled throughthe support 12, epoxy POSS resin film 14, 14′, 14″, and patternedhydrophobic polymer layer 16. Alternatively, the layers on the support12 may be removed from those regions where it is desirable to form aport and/or bonding region. The layers may be removed prior to portdrilling and bonding. By connecting to ports, the reaction automationdevice may control the flow of reagent(s) and product(s) in the sealedflow channels. The reaction automation device may, in some applications,adjust the pressure, temperature, gas composition and otherenvironmental conditions of the flow cell. Further, in someapplications, ports may be drilled in the top substrate or in both thetop substrate and through the support 12, epoxy POSS resin film 14, 14′,14″, and patterned hydrophobic polymer layer 16. In some applications,the reactions taking place in sealed flow channels may be monitoredthrough the top substrate by imaging or measurements of heat, lightemission and/or fluorescence.

Additional Notes

It should be appreciated that all combinations of the foregoing concepts(provided such concepts are not mutually inconsistent) are contemplatedas being part of the inventive subject matter disclosed herein. Inparticular, all combinations of claimed subject matter appearing at theend of this disclosure are contemplated as being part of the inventivesubject matter disclosed herein. It should also be appreciated thatterminology explicitly employed herein that also may appear in anydisclosure incorporated by reference should be accorded a meaning mostconsistent with the particular concepts disclosed herein.

All publications, patents, and patent applications cited in thisSpecification are hereby incorporated by reference in their entirety.

Reference throughout the specification to “one example”, “anotherexample”, “an example”, and so forth, means that a particular element(e.g., feature, structure, and/or characteristic) described inconnection with the example is included in at least one exampledescribed herein, and may or may not be present in other examples. Inaddition, it is to be understood that the described elements for anyexample may be combined in any suitable manner in the various examplesunless the context clearly dictates otherwise.

It is to be understood that the ranges provided herein include thestated range and any value or sub-range within the stated range. Forexample, a range from about 10 kDa to about 1500 kDa, should beinterpreted to include not only the explicitly recited limits of fromabout 10 kDa to about 1500 kDa, but also to include individual values,such as about 88 kDa, about 325 kDa, about 425 kDa, about 975.5 kDa,etc., and sub-ranges, such as from about 25 kDa to about 900 kDa, fromabout 335 kDa to about 680 KDa, etc. Furthermore, when “about” isutilized to describe a value, this is meant to encompass minorvariations (up to +/−10%) from the stated value.

While several examples have been described in detail, it is to beunderstood that the disclosed examples may be modified. Therefore, theforegoing description is to be considered non-limiting.

What is claimed is:
 1. A method, comprising: forming a patternedhydrophobic polymer layer on a cross-linked epoxy polyhedral oligomericsilsesquioxane (POSS) resin film on a support surface, thereby exposingdiscrete areas of the cross-linked epoxy POSS resin film; applying apolymer coating to form an attached coating portion on the exposeddiscrete areas and an unattached coating portion on the patternedhydrophobic layer; and washing the unattached coating portion off of thepatterned hydrophobic layer.
 2. The method as defined in claim 1,further comprising forming the cross-linked epoxy POSS resin film on thesupport surface, the forming involving: mixing an epoxy silane and atleast one epoxy POSS monomeric unit in the presence of a photoacidgenerator and optionally a sensitizer to form a resin precursor;depositing the resin precursor on the support surface; and curing theresin precursor to form the cross-linked epoxy POSS resin film.
 3. Themethod as defined in claim 2, wherein the mixing and depositing occur atthe same time.
 4. The method as defined in claim 2, wherein the epoxysilane is an epoxy silane bound to the support surface.
 5. The method asdefined in claim 1, further comprising forming the cross-linked epoxyPOSS resin film on the support surface, the forming involving: mixing anepoxy silane, epoxycyclohexylalkyl POSS, and glycidyl POSS in thepresence of a photoacid generator and optionally a sensitizer to form aresin precursor; depositing the resin precursor on the support surface;and curing the resin precursor to form the cross-linked epoxy POSS resinfilm.
 6. The method as defined in claim 1, wherein prior to forming thepatterned hydrophobic polymer layer, the method further comprises:exposing the cross-linked epoxy POSS resin film to plasma ashing or achemical treatment to introduce —OH groups to the cross-linked epoxyPOSS resin film; and attaching functional groups to at least some of the—OH groups, the functional groups being selected from the groupconsisting of:

wherein n ranges from 1 to 20, and wherein - - - represents analkylsilane, a poly(ethylene glycol)-silane, an alkyl, or a polyethyleneglycol chain.
 7. The method as defined in claim 1, further comprisingforming the cross-linked epoxy POSS resin film on the support surface,the forming involving: mixing, in the presence of a photoacid generatorand optionally a sensitizer, an epoxy silane, epoxycyclohexylalkyl POSS,glycidyl POSS, and a POSS core including at least one epoxy functionalgroup and a non-epoxy functional group to form a resin precursor;depositing the resin precursor on the support surface; and curing theresin precursor to form the cross-linked epoxy POSS resin film.
 8. Themethod as defined in claim 1, wherein washing involves sonication inwater.
 9. The method as defined in claim 1, wherein forming thepatterned hydrophobic polymer layer involves: i) depositing ahydrophobic polymer on the cross-linked epoxy POSS resin film; andpatterning the deposited hydrophobic polymer using at least one ofnanoimprint lithography and photolithography; or ii) depositing thehydrophobic polymer in a pattern on the cross-linked epoxy POSS resinfilm using at least one of inkjet printing and microcontact printing.10. The method as defined in claim 1, further comprising grafting anamplification primer to the attached coating portion.
 11. A method,comprising: forming a cross-linked epoxy POSS resin film on a supportsurface, where the forming involves mixing a support-bound epoxy silanewith one or more epoxy-functionalized POSS reagents in the presence of aphotoacid generator and optionally a sensitizer to form a support-boundresin precursor; curing the resin precursor to form a support-boundcross-linked epoxy POSS resin film; and forming a hydrophobic polymerlayer on the cross-linked, support-bound epoxy POSS resin film, whereinthe hydrophobic polymer layer is patterned to expose the cross-linked,support-bound epoxy POSS resin film in discrete areas or wells whileremaining on the cross-linked, support-bound epoxy POSS resin film ininterstitial areas of the cross-linked, support-bound epoxy POSS resinfilm between the discrete areas or wells.
 12. The method as defined inclaim 11, wherein the one or more epoxy-functionalized POSS reagentscomprise an epoxycyclohexylalkyl POSS and a glycidyl POSS.
 13. Themethod as defined in claim 11, further comprising reacting the supportsurface with an epoxy silane to form the support-bound epoxy silane.